Direct Inoculum Protocol (DIP) for Bacterial Identification Using an Automated Microbial Identification and Antimicrobial Susceptibility Testing System (aMIAST)

To begin, swab the septum of an automated blood culture bottle, having monomorphic gram-negative organisms with 70%isopropyl alcohol inside a class 2A biosafety cabinet. Draw five milliliters of blood broth mixture into a sterile syringe equipped with a 21 gauge needle. Next, disinfect the rubber septum of a rubber separator tube.

Transfer the blood broth mixture into the tube. Centrifuge the sample at 160 G for 10 minutes. Now, carefully pipette out the supernatant inside a biosafety cabinet.

Transfer the supernatant to a new plain blood collection vial. Place the capped vial in a centrifuge at 2000 G for 10 minutes. Now, aspirate the supernatant with a sterile pipette.

Then dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle. Using a sterile inoculation loop, pick the bacterial pellet from the bottom of the vial and inoculate it into the aMIAST tube. Use sterile saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63.

Then place a capillary attachment of the gram-negative aMIAST identification card in the first tube. Position the selected cards appropriately on the cassette. Now, load the cassette into its position in the filler chamber with the sample barcode facing inward.

Close the door and press fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now, transfer the cassette from the filling chamber into the loading chamber.

Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The results of seven positive blood culture bottles, identified using the direct inoculum protocol, were discordant until the species level.

Of the 105 RAST reportable gram negatives identified by the standard inoculum protocol, 98 were correctly identified by the direct inoculum protocol. Among 99 non-RAST reportable gram negatives, a RAST reportable gram negative was identified in four positive blood culture bottles.